The activities of the U.S. Department of Defense (DoD) and its contractors result in the generation of large amounts of hazardous wastes. Many of the constituents of concern are waterborne or have become waterborne as a result of leaks or spills. Among the most troublesome of these wastes are organic solvents. Even at low concentrations, these constituents are often toxic, tend to be resistent to conventional treatment methods and are persistent in the environment. Common halogenated hydrocarbon waste constituents include the following:
tetrachloroethylene (PCE) PA1 trichloroethylene (TCE) PA1 1,1,1-trichloroethane (TCA)
Halogenated hydrocarbons are used as solvents and in vapor degreasing operations.
Prior art processes have been developed that accomplish microbial dehalogenation of halogenated hydrocarbons. Processes for dechlorination of chlorinated aliphatic hydrocarbons by aerobic microorganisms are disclosed in U.S. Pat. Nos. 4,877,736, 4,959,315, and 5,024,949, the disclosures of which patents are incorporated by reference herein as if fully set forth.
Because of the significant advantages of accomplishing dehalogenation under anaerobic conditions, many researchers have investigated methods involving anaerobic reductive dehalogenation. Reductive dehalogenation is an oxidation-reduction reaction that results in the replacement of a halogen atom with a hydrogen atom. See Egli, Tschan, Scholtz, Cook and Leisinger in Applied and Environmental Microbiology, Vol. 54, pp. 2819-2824 (1988); Fathepure, Tiedje and Boyd in Applied and Environmental Microbiology, Vol. 54, pp. 327-330 (1988) and Freedman and Gossett in Applied and Environmental Microbiology, Vol. 55, pp. 2144-2151 (1989).
Different methanogenic bacteria strains vary in their ability to reductively dechlorinate chlorinated aliphatic hydrocarbons. Methanosarcina mazei strain S6 and Methanosarcina sp. strain DCM isolated from a methanogenic enrichment growing on chlorophenol can produce TCE from PCE while Methanosarcina acetivorans and a highly enriched culture of Methanothrix sp. also obtained from the chlorophenol enrichment do not have this ability. See Fathepure and Boyd in Applied and Environmental Microbiology, Vol. 54, pp. 2976-2980 (1988); Fathepure and Boyd in FEMS Microbiology Letters, Vol. 49, pp. 149-156 (1988) and Fathepure, Nengu and Boyd in Applied and Environmental Microbiology, Vol. 53, pp. 267114 2674 (1987). Methanobacterium thermoautotrophicum can produce ethylene (ethene) from 1,2-dichloroethylene (1,2-dichloroethene). See Egli, Scholtz, Cook and Leisinger in FEMS Microbiology Letters, Vol. 43, pp. 257-261 (1987). Methanobacterium thermoautotrophicum can also dechlorinate carbon tetrachloride (tetrachloromethane). See Egli, Stromeyer, Cook and Leisinger in FEMS Microbiology Letters, Vol. 68, pp. 207-212 (1990). Methanobacterium thermoautotrophicum strain AH, Methanococcus deltae strain ALH and Methanococcus thermolithotrophicus can produce ethane from bromoethane, ethylene from bromoethane sulfonate, 1,2-dibromoethane and 1,2-dichloroethane and acetylene from 1,2-dibromoethylene. See Belay and Daniels in Applied and Environmental Microbiology, Vol. 53, pp. 1604-1610 (1987).
Krone, Laufer, Thauer and Hogenkamp in Biochemistry, Vol. 28, pp. 10061-10065 (1989) disclosed that purified coenzyme F.sub.430 obtained from Methanobacterium thermoautotropicum strain Marburg grown on H.sub.2 --CO.sub.2 catalyzed the dechlorination of carbon tetrachloride (CCl.sub.4) to form chloroform (CHCl.sub.3) methylene chloride (CH.sub.2 Cl.sub.2), methyl chloride (CH.sub.3 Cl), methane (CH.sub.4) and small amounts of ethane (CH.sub.3 CH.sub.3) with titanium (III) citrate as electron donor. Coenzyme F.sub.430 also catalyzed the reduction of chloroform, methylene chloride and methyl chloride individually.
Concerning the dehalogenation ability of methanogens of the genus Methanosarcina, the prior art teaches that only three strains, namely Methanosarcina mazei strain S6, Methanosarcina sp. strain DCM, and Methanosarcina barkeri strain Fusaro have the ability to dehalogenate as whole cells. For example, Fathepure and Boyd in FEMS Microbiology Letters, Vol. 49, pp. 149-156 (1988) stated "The dechlorination of PCE by our Methanosarcina sp. appears to be a unique characteristic as other acetate-utilizing methanogens show either significantly lower or no activity against PCE." Acceptance of these teachings is illustrated by use of pure cultures of these Methanosarcina strains in research documenting their ability to dehalogenate carbon tetrachloride, chloroform and bromoform. See Mikesell and Boyd in Applied and Environmental Microbiology, Vol. 56, pp. 1198-1201 (1990). Methanosarcina sp. strain DCM has been shown not to be the same bacterium as Methanosarcina barkeri strain 227 (ATCC 43241). See Fathepure and Boyd in Applied and Environmental Microbiology, Vol. 54, pp. 2976-2980 (1988).
Jablonski and Ferry in FEMS Microbiology Letters, Vol. 96, pp. 55-60 (1992) disclosed that purified carbon monoxide dehydrogenase enzyme complex obtained from Methanosarcina thermophila dechlorinated TCE in the presence of either carbon monoxide or titanium (III) citrate as a reductant. About one-third of the TCE was transformed to cis 1,2-dichloroethylene (predominantly), trans 1,2-dichloroethylene, vinyl chloride, ethylene and (traces of) 1,1-dichloroethylene.
Krone, Laufer, Thauer and Hogenkamp in Biochemistry, Vol. 28, pp. 10061-10065 (1989) disclosed that cell suspensions of Methanosarcina barkeri strain Fusaro (ATCC 29787) harvested from a methanol or acetate medium could dehalogenate carbon tetrachloride to form chloroform, methylene chloride and minor amounts of methyl chloride with carbon monoxide as the electron donor. This strain was also able to dehalogenate chloroform and methylene chloride individually under the same conditions. The strain was unable to accomplish the dehalogenation of methyl chloride.
Krone and Thauer in FEMS Microbiology Letters, Vol. 90, pp. 201-204 (1992) disclosed that cell suspensions of Methanosarcina barkeri strain Fusaro (ATCC 29787) harvested from a methanol medium were able to catalyze the reductive dehalogenation of trichlorofluoromethane (CFC-11, also known as FREON 11) to form CHFCl.sub.2, CO and fluoride and minute amounts of CH.sub.2 FCl in the presence of either H.sub.2 or CO. The presence of either H.sub.2 or CO was necessary for dehalogenation of CFC-11 to occur. Dehalogenation of CFC-12 (dichlorodifluoromethane) occurred at less than 5 percent of the rate at which CFC-11 was dehalogenated. CFC-11 completely inhibited methanogenesis in Methanosarcina barkeri strain Fusaro at the concentrations tested (6.7 .mu.mol in a 120-ml serum bottle containing 10 ml of cell suspension).
In summary, the prior art teaches that some species of the genus Methanosarcina (e.g., Methanosarcina acetivorans) are unable to dehalogenate halogenated hydrocarbons such as PCE. It also teaches that, in the genus Methanosarcina, only Methanosarcina sp. strain DCM, Methanosarcina mazei strain S6 and Methanosarcina barkeri strain Fusaro possess dehalogenation ability as whole cells. The prior art teaches that the ability for a bacterial strain to catalyze a dehalogenation reaction varies not only with the halogenated hydrocarbon (electron acceptor) but also with the presence or absence of a specific reducing agent (electron donor). No prior art teaches or suggests that Methanosarcina barkeri strain 227 (ATCC 43241) or that any strain of Methanosarcina vacuolata can dechlorinate any halogen. No prior art teaches or suggests that any methanogen can dechlorinate TCA during heterotrophic growth.
Methanogenic bacteria are archaeobacteria that belong to one of the orders Methanobacteriales, Methanococcales, or Methanomicrobiales. The family Methanosarcinacae is one of two families in the order Methanobacteriales. The family Methanosarcinacae includes three genera: Methanosarcina, Methanolobus and Methanothrix.
Methanosarcina barkeri is a species of the genus Methanosarcina. Its energy-yielding metabolism involves methane production. During autotrophic metabolism, H.sub.2 --CO.sub.2, and during heterotrophic metabolism, methanol, (mono)methylamine, dimethylamine, trimethylamine and acetate are reduced to CH.sub.4 without intermediate oxidation to CO.sub.2. Growth and CH.sub.4 formation are more rapid in media with H.sub.2 --CO.sub.2 or methanol as substrates than with acetate Cells of Methanosarcina barkeri do not contain gas vesicles. Optimum growth is obtained at pH 7.0 and at 30.degree.-40.degree. C.
Four strains of the methanogen Methanosarcina barkeri are available from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852-1776. Their strain names and accession numbers from the ATCC and the Deutsche Sammulung yon Mikroorganismen (DSM), Gottingen, Federal Republic of Germany are as follows:
______________________________________ Accession number Strain name ATCC DSM ______________________________________ 3 29786 805 Fusaro 29787 804 DM 29894 -- FA.sup.r 9 43240 -- 227 43241 1538 ______________________________________
Information on these strains and their growth requirements is available in the literature. See Hippe, Caspari, Fiebig and Gottschalk in Proceedings of the National Academy of Science USA, Vol. 76, pp. 494-498 (1979); Mah, Smith and Baresi in Applied and Environmental Microbiology, Vol. 35, pp. 1174-1184 (1978); Smith and Lequerica in Journal of Bacteriology, Vol. 164, pp. 618-625 (1985); Scherer and Sahm in European Journal of Applied Microbiology and Biotechnology, Vol. 12, pp. 28-35 (1981); Kandler and Hippe in Archives of Microbiology, Vol. 113, pp. 57-60 (1977) and Staley, Bryant, Pfennig and Holt (eds.) in Bergey's Manual of Systematic Bacteriology, Vol. 3, Baltimore: Williams & Wilkins (1989).
Methanosarcina vacuolata is also a species of the genus Methanosarcina. H.sub.2 --CO.sub.2, methanol, methylamines and acetate may be used as substrates. Growth on methanol is faster than on acetate. The cells of Methanosarcina vacuolata may contain gas vesicles. Optimum growth is obtained at pH 7.5 and 37.degree.-40.degree. C.
One strain of the methanogen Methanosarcina vacuolata is available from the ATCC. The strain name and accession numbers from the ATCC and the DSM are as follows:
______________________________________ Accession number Strain name ATCC DSM ______________________________________ Z-761 35090 1232 ______________________________________
Information on this strain is available in the literature. See Zhilina and Zavarzin in Mikrobiologiya, Vol. 48, pp. 279-285 (1979); Zhilina and Zavarzin in International Journal of Systematic Bacteriology, Vol. 37, pp. 281-283 (1987) and Staley, Bryant, Pfennig and Holt (eds.) in Bergey's Manual of Systematic Bacteriology, Vol. 3, Baltimore: Williams & Wilkins (1989).